Herpes simplex virus (HSV) remains a ubiquitous human pathogen responsible for serious disease outcomes, including blindness, fatal encephalitis, and increased risk for HIV, dementia, heart disease, and diabetes. Upon primary infection, virus enters epithelial cells which support replication, local spread and dissemination into the peripheral and central the nervous systems. In the nervous system, HSV has evolved a complex regulatory strategy that protects the genetically diverse host population from lethal infection, preserves a large repository of latent viral genomes, and supports periodic activation of the lytic cycle in rare neurons, sufficient to infect new hosts. How this balance between lytic and latent is achieved is central to our goal to develop strategies to prevent reactivation associated disease and transmission. Approaches we developed have yielded major insight into the role of de novo expression VP16 in pro-lytic regulation of HSV in the nervous system. Importantly dysregulation of this arm of the network results in uncontrolled replication in the nervous system and death despite immune competency. Using a similar strategy we have now identified a counterbalancing latency promoting non-coding RNA component that, when dysregulated, has the capacity to fully block viral reactivation in vivo. Viral mutants in which a 1.6 kb non-coding DNA fragment from upstream and on the opposite strand of the latency associated transcript locus (LAT) is ectopically expressed from the LAT promoter replicate like wild type (wt) in mouse eyes and trigeminal ganglia (TG), establish latency efficiently, but fail t exit latency following stress. These mutants display a >100-fold reduction in virulence, and when co-infected even quench the virulence of highly neurovirulent HSV-1 strain McKrae and HSV-2 strain 186. Expression of the fragment in the other orientation does not alter viral properties. We hypothesize that the extreme phenotypes displayed are the result of dysregulated riboregulator expression and that they will permit us to deduced the normal targets of the effector molecules. In this R21 application we propose to identify the molecular networks perturbed by the riboregulators(s), determine its primary targets and identify of the riboregulator itself (likely miR-H6) and characterize its normal pattern of expression.